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Abstract:

The present invention relates generally to identification of HPV, and
provides methods, compositions and kits useful for this purpose when
combined, for example, with molecular mass or base composition analysis.

Claims:

1. A composition comprising at least one purified oligonucleotide primer
pair that comprises forward and reverse primers, wherein said primer pair
comprises nucleic acid sequences that are substantially complementary to
nucleic acid sequences of two or more different bioagents belonging to
the HPV family, wherein said primer pair is configured to produce
amplicons comprising different base compositions that correspond to said
two or more different bioagents.

2. The composition of claim 1, wherein said primer pair is configured to
hybridize with conserved regions of said two or more different bioagents
and flank variable regions of said two or more different bioagents.

3. The composition of claim 1, wherein said forward and reverse primers
are about 15 to 35 nucleobases in length, and wherein the forward primer
comprises at least 70% sequence identity with a sequence selected from
the group consisting of SEQ ID NOS: 1-8, and the reverse primer comprises
at least 70% sequence identity with a sequence selected from the group
consisting of SEQ ID NOS: 9-16.

5. The composition of claim 1, wherein said forward and reverse primers
are about 15 to 35 nucleobases in length, and wherein: the forward primer
comprises at least 70%, sequence identity with the sequence of SEQ ID NO:
1, and the reverse primer comprises at least 70% sequence identity with
the sequence of SEQ ID NO: 9; the forward primer comprises at least 70%
sequence identity with the sequence of SEQ ID NO: 2, and the reverse
primer comprises at least 70% sequence identity with the sequence of SEQ
ID NO: 10; the forward primer comprises at least 70% sequence identity
with the sequence of SEQ ID NO: 3, and the reverse primer comprises at
least 70% sequence identity with the sequence of SEQ ID NO: 11; the
forward primer comprises at least 70% sequence identity with the sequence
of SEQ ID NO: 4, and the reverse primer comprises at least 70% sequence
identity with the sequence of SEQ ID NO: 12; the forward primer comprises
at least 70% sequence identity with the sequence of SEQ ID NO: 5, and the
reverse primer comprises at least 70% sequence identity with the sequence
of SEQ ID NO: 13; the forward primer comprises at least 70% sequence
identity with the sequence of SEQ ID NO: 6, and the reverse primer
comprises at least 70% sequence identity with the sequence of SEQ ID NO:
14; the forward primer comprises at least 70% sequence identity with the
sequence of SEQ ID NO: 7, and the reverse primer comprises at least 70%
sequence identity with the sequence of SEQ ID NO: 15; and the forward
primer comprises at least 70% sequence identity with the sequence of SEQ
ID NO: 8, and the reverse primer comprises at least 70% sequence identity
with the sequence of SEQ ID NO: 16.

6. The composition of claim 1, wherein said different base compositions
identify said two or more different bioagents at genus, species, or
sub-species levels.

7. The composition of claim 1, wherein said two or more amplicons are 45
to 200 nucleobases in length.

8. A kit comprising the composition of claim 1.

9. The composition of claim 1, wherein said different bioagents are
selected from the group consisting of HPV-16, HPV-31, HPV-18, HPV-45,
HPV-6, and HPV-11.

10. The composition of claim 1, wherein a non-templated T residue on the
5'-end of said forward and/or reverse primer is removed.

11. The composition of claim 1, wherein said forward and/or reverse
primer further comprises a non-templated T residue on the 5'-end.

14. The composition of claim 13, wherein said modified nucleobase is
5-propynyluracil or 5-propynylcytosine.

15. The composition of claim 13, wherein said modified nucleobase is a
mass modified nucleobase.

16. The composition of claim 15, wherein said mass modified nucleobase is
5-Iodo-C.

17. The composition of claim 13, wherein said modified nucleobase is a
universal nucleobase.

18. The composition of claim 17, wherein said universal nucleobase is
inosine.

19. A composition comprising an isolated primer 15-35 bases in length
selected from the group consisting of SEQ ID NOs 1-16.

20. A kit, comprising at least one purified oligonucleotide primer pair
that comprises forward and reverse primers that are about 20 to 35
nucleobases in length, and wherein said forward primer comprises at least
70% sequence identity with a sequence selected from the group consisting
of SEQ ID NOS: 1-8, and said reverse primer comprises at least 70%
sequence identity with a sequence selected from the group consisting of
SEQ ID NOS: 9-16.

21. A method of determining the presence of a HPV in at least one sample,
the method comprising: (a) amplifying one or more segments of at least
one nucleic acid from said sample using at least one purified
oligonucleotide primer pair that comprises forward and reverse primers
that are about 20 to 35 nucleobases in length, and wherein said forward
primer comprises at least 70% sequence identity with a sequence selected
from the group consisting of SEQ ID NOs: 1-8, and said reverse primer
comprises at least 70% sequence identity with a sequence selected from
the group consisting of SEQ ID NOs: 9-16 to produce at least one
amplification product; and (b) detecting said amplification product,
thereby determining said presence of said HPV in said sample.

22. The method of claim 21, wherein (a) comprises amplifying said one or
more segments of said at least one nucleic acid from at least two samples
obtained from different geographical locations to produce at least two
amplification products, and (b) comprises detecting said amplification
products, thereby tracking an epidemic spread of said HPV.

23. The method of claim 21, wherein (b) comprises determining an amount
of said HPV in said sample.

25. The method of claim 21, wherein (b) comprises determining a base
composition of said amplification product, wherein said base composition
identifies the number of A residues, C residues, T residues, G residues,
U residues, analogs thereof and/or mass tag residues thereof in said
amplification product, whereby said base composition indicates the
presence of HPV in said sample or identifies said HPV in said sample.

26. The method of claim 25, comprising comparing said base composition of
said amplification product to calculated or measured base compositions of
amplification products of one or more known HPV present in a database
with the proviso that sequencing of said amplification product is not
used to indicate the presence of or to identify said HPV, wherein a match
between said determined base composition and said calculated or measured
base composition in said database indicates the presence of or identifies
said HPV.

27. A method of identifying one or more HPV bioagents in a sample, the
method comprising: (a) amplifying two or more segments of a nucleic acid
from said one or more HPV bioagents in said sample with two or more
oligonucleotide primer pairs to obtain two or more amplification
products; (b) determining two or more molecular masses and/or base
compositions of said two or more amplification products; and (c)
comparing said two or more molecular masses and/or said base compositions
of said two or more amplification products with known molecular masses
and/or known base compositions of amplification products of known HPV
bioagents produced with said two or more primer pairs to identify said
one or more HPV bioagents in said sample.

28. The method of claim 27, comprising identifying said one or more HPV
bioagents in said sample using three, four, five, six, seven, eight or
more primer pairs.

29. The method of claim 27, wherein said one or more HPV bioagents in
said sample cannot be identified using a single primer pair of said two
or more primer pairs.

30. The method of claim 27, comprising obtaining said two or more
molecular masses of said two or more amplification products via mass
spectrometry.

31. The method of claim 27, comprising calculating said two or more base
compositions from said two or more molecular masses of said two or more
amplification products.

32. The method of claim 27, wherein said HPV bioagents are selected from
the group consisting of a Papillomaviridae family, a genus thereof; a
species thereof, a sub-species thereof, and combinations thereof.

33. The method of claim 27, wherein said two or more primer pairs
comprise two or more purified oligonucleotide primer pairs that each
comprise forward and reverse primers that are about 20 to 35 nucleobases
in length, and wherein said forward primers comprise at least 70%
sequence identity with a sequence selected from the group consisting of
SEQ ID NOS: 1-8, and said reverse primers comprise at least 70% sequence
identity with a sequence selected from the group consisting of SEQ ID.
NOS: 9-16 to obtain an amplification product.

35. The method of claim 27, wherein said determining said two or more
molecular masses and/or base compositions is conducted without sequencing
said two or more amplification products.

36. The method of claim 27, wherein said one or more HPV bioagents in
said sample cannot be identified using a single primer pair of said two
or more primer pairs.

37. The method of claim 27, wherein said one or more HPV bioagents in a
sample are identified by comparing three or more molecular masses and/or
base compositions of three or more amplification products with a database
of known molecular masses and/or known base compositions of amplification
products of known HPV bioagents produced with said three or more primer
pairs.

38. The method of claim 27, wherein said two or more segments of said
nucleic acid are amplified from a single gene.

39. The method of claim 27, wherein said two or more segments of said
nucleic acid are amplified from different genes.

40. The method of claim 27, wherein members of said primer pairs
hybridize to conserved regions of said nucleic acid that flank a variable
region.

41. The method of claim 40, wherein said variable region varies between
at least two of said HPV bioagents.

42. The method of claim 40, wherein said variable region uniquely varies
between at least five of said HPV bioagents.

43. The method of claim 27, wherein said two or more amplification
products obtained in (a) comprise major classification and subgroup
identifying amplification products.

44. The method of claim 43, comprising comparing said molecular masses
and/or said base compositions of said two or more amplification products
to calculated or measured molecular masses or base compositions of
amplification products of known HPV bioagents in a database comprising
genus specific amplification products, species specific amplification
products, strain specific amplification products or nucleotide
polymorphism specific amplification products produced with said two or
more oligonucleotide primer pairs, wherein one or more matches between
said two or more amplification products and one or more entries in said
database identifies said one or more HPV bioagents, classifies a major
classification of said one or more HPV bioagents, and/or differentiates
between subgroups of known and unknown HPV bioagents in said sample.

45. The method of claim 44, wherein said major classification of said one
or more HPV bioagents comprises a genus or species classification of said
one or more HPV bioagents.

46. The method of claim 44, wherein said subgroups of known and unknown
HPV bioagents comprise family, strain and nucleotide variations of said
one or more HPV bioagents.

47. A system, comprising: (a) a mass spectrometer configured to detect
one or more molecular masses of amplicons produced using at least one
purified oligonucleotide primer pair that comprises forward and reverse
primers, wherein said primer pair comprises nucleic acid sequences that
are substantially complementary to nucleic acid sequences of two or more
different HPV bioagents; and (b) a controller operably connected to said
mass spectrometer, said controller configured to correlate said molecular
masses of said amplicons with one or more HPV bioagent identities.

48. The system of claim 47, wherein said HPV bioagent identities are at
genus, species, and/or sub-species levels.

49. The system of claim 47, wherein said forward and reverse primers are
about 15 to 35 nucleobases in length, and wherein the forward primer
comprises at least 70% sequence identity with a sequence selected from
the group consisting of SEQ ID NOS: 1-8, and the reverse primer comprises
at least 70% sequence identity with a sequence selected from the group
consisting of SEQ ID NOS: 9-16.

50. The system of claim 47, wherein said primer pair is selected from the
group of primer pair sequences consisting of: SEQ ID NOS: 1:9, 2:10,
3:11, 4:12, 5:13, 6:14, 7:15, and 8:16.

51. The system of claim 47, wherein said controller is configured to
determine base compositions of said amplicons from said molecular masses
of said amplicons, which base compositions correspond to said one or more
HPV bioagent identities.

52. The system of claim 47, wherein said controller comprises or is
operably connected to a database of known molecular masses and/or known
base compositions of amplicons of known HPV bioagents produced with the
primer pair.

53. A purified oligonucleotide primer pair, comprising a forward primer
and a reverse primer that each independently comprise 14 to 40
consecutive nucleobases selected from the primer pair sequences shown in
Table 1 and/or Table 2, which primer pair is configured to generate an
amplicon between about 50 and 150 consecutive nucleobases in length.

Description:

CROSS REFERENCE TO RELATED PATENT APPLICATIONS

[0001] The present application claims the benefit of priority to U.S.
Provisional Application No. 61/102,324, filed Oct. 2, 2008 and is further
a continuation-in-part of U.S. patent application Ser. No. 11/368,233,
filed Mar. 3, 2006, which claims the benefit of priority to U.S.
Provisional Application Nos. 60/658,248, filed Mar. 3, 2005, 60/705,631,
filed Aug. 3, 2005, 60/732,539, filed Nov. 1, 2005, and 60/740,617, filed
Nov. 28, 2005, which are each incorporated by reference in their
entirety.

FIELD OF THE INVENTION

[0003] The present invention relates generally to identification of Human
papillomavirus (HPV), and provides methods, compositions and kits useful
for this purpose when combined, for example, with molecular mass or base
composition analysis.

BACKGROUND OF THE INVENTION

[0004] Papillomaviruses are a diverse group of DNA-based viruses that
infect the skin and mucous membranes of humans and a variety of animals.
Approximately 130 HPV types have been identified. About 30-40 HPV types
are typically transmitted through sexual contact and infect the
anogenital region. Different HPV types are associated with different
pathological risks. Some HPV types result in latent infection, while some
can cause warts, and others may cause a subclinical infection resulting
in precancerous lesions. Persistent infection with a "high-risk" subset
of sexually transmitted HPV may lead to potentially precancerous lesions
and can progress to invasive cancer. HPV infection is a necessary factor
in the development of nearly all cases of cervical cancer.

SUMMARY OF THE INVENTION

[0005] The present invention relates generally to detection and
identification of HPV, and provides methods, compositions and kits useful
for this purpose when combined, for example, with molecular mass or base
composition analysis. However, the compositions and methods find use in a
variety of biological sample analysis techniques and are not limited to
processes that employ or require molecular mass or base composition
analysis. For example, primers described herein find use in a variety of
research, surveillance, and diagnostic approaches that utilize one or
more primers, including a variety of approaches that employ the
polymerase chain reaction.

[0006] Ito further illustrate, in certain embodiments, the invention for
the rapid detection and characterization of papillomavirus. In some
embodiments the present invention provides a composition comprising at
least one purified oligonucleotide primer pair that comprises forward and
reverse primers, wherein said primer pair comprises nucleic acid
sequences that are substantially complementary to nucleic acid sequences
of two or more different bioagents belonging to the Papillomaviridae
family, wherein the primer pair is configured to produce amplicons
comprising different base compositions that correspond to the two or more
different bioagents. In addition to compositions and kits that include
one or more of the primer pairs described herein, the invention also
relates to methods and systems.

[0008] In another aspect, the invention provides a purified
oligonucleotide primer pair, comprising a forward primer and a reverse
primer that each independently comprise 14 to 40 consecutive nucleobases
selected from the primer pair sequences shown in Table 1 and/or Table 2,
which primer pair is configured to generate an amplicon between about 50
and 150 consecutive nucleobases in length.

[0009] In another aspect, the invention provides a kit comprising at least
one purified oligonucleotide primer pair that comprises forward and
reverse primers that are about 20 to 35 nucleobases in length, and
wherein the forward primer comprises at least 70%, at least 80%, at least
90%, at least 95%, or at least 100% sequence identity with a sequence
selected from the group consisting of SEQ ID NOS: 1-8 and 17-43, and the
reverse primer comprises at least 70% sequence identity (e.g., 75%, 85%,
or 95%) with a sequence selected from the group consisting of SEQ ID NOS:
9-16 and 44-70. In some embodiments, the kit comprises a primer pair that
is a broad range survey primer pair (e.g., specific for nucleic acid of a
housekeeping gene found in many or all members of a category of
organism).

[0010] In other embodiments, the amplicons produced with the primers are
45 to 200 nucleobases in length (e.g., 45 . . . 75 . . . 125 . . . 175 .
. . 200). In some embodiments, a non-templated T residue on the 5'-end of
said forward and/or reverse primer is removed. In still other
embodiments, the forward and/or reverse primer further comprises a
non-templated T residue on the 5'-end. In additional embodiments, the
forward and/or reverse primer comprises at least one molecular mass
modifying tag. In some embodiments, the forward and/or reverse primer
comprises at least one modified nucleobase. In further embodiments, the
modified nucleobase is 5-propynyluracil or 5-propynylcytosine. In other
embodiments, the modified nucleobase is a mass modified nucleobase. In
still other embodiments, the mass modified nucleobase is 5-Iodo-C. In
additional embodiments, the modified nucleobase is a universal
nucleobase. In some embodiments, the universal nucleobase is inosine. In
certain embodiments, kits comprise the compositions described herein.

[0011] In particular embodiments, the present invention provides methods
of determining a presence of an HPV in at least one sample, the method
comprising: (a) amplifying one or more (e.g., two or more, three or more,
four or more, etc.; one to two, one to three, one to four, etc.; two,
three, four, etc.) segments of at least one nucleic acid from the sample
using at least one purified oligonucleotide primer pair that comprises
forward and reverse primers that are about 20 to 35 nucleobases in
length, and wherein the forward primer comprises at least 70% (e.g., 70%
. . . 75% . . . 90% . . . 95% . . . 100%) sequence identity with a
sequence selected from the group consisting of SEQ ID NOs: 1-8 and 17-43,
and the reverse primer comprises at least 70% (e.g., 70% . . . 75% . . .
90% . . . 95% . . . 100%) sequence identity with a sequence selected from
the group consisting of SEQ ID NOs: 9-16 and 44-70 to produce at least
one amplification product; and (b) detecting the amplification product,
thereby determining the presence of the HPV in the sample.

[0012] In certain embodiments, step (b) comprises determining an amount of
the HPV in the sample. In further embodiments, step (b) comprises
detecting a molecular mass of the amplification product. In other
embodiments, step (b) comprises determining a base composition of the
amplification product, wherein the base composition identifies the number
of A residues, C residues, T residues, G residues, U residues, analogs
thereof and/or mass tag residues thereof in the amplification product,
whereby the base composition indicates the presence of the HPV in the
sample or identifies the pathogenicity of the HPV in the sample. In
particular embodiments, the methods further comprise comparing the base
composition of the amplification product to calculated or measured base
compositions of amplification products of one or more known HPV present
in a database, for example, with the proviso that sequencing of the
amplification product is not used to indicate the presence of or to
identify the HPV, wherein a match between the determined base composition
and the calculated or measured base composition in the database indicates
the presence of or identifies the HPV. In some embodiments, the
identification of HPV is at the biological kingdom level, phylum level,
class level, order level, family level, genus level, species level,
sub-type level (e.g., stain level), genotype level, or individual
identity level.

[0013] In some embodiments, the present invention provides methods of
identifying one or more HPV bioagents in a sample, the method comprising:
amplifying two or more segments of a nucleic acid from the one or more
HPV bioagents in the sample with two or more oligonucleotide primer pairs
to obtain two or more amplification products (e.g., from a single
bioagent); (b) determining two or more molecular masses and/or base
compositions of the two or more amplification products; and (c) comparing
the two or more molecular masses and/or the base compositions of the two
or more amplification products with known molecular masses and/or known
base compositions of amplification products of known HPV bioagents
produced with the two or more primer pairs to identify the one or more
HPV bioagents in the sample. In certain embodiments, the methods comprise
identifying the one or more HPV bioagents in the sample using three,
four, five, six, seven, eight or more primer pairs. In other embodiments,
the one or more HPV bioagents in the sample cannot be identified using a
single primer pair of the two or more primer pairs. In particular
embodiments, the methods comprise obtaining the two or more molecular
masses of the two or more amplification products via mass spectrometry.
In certain embodiments, the methods comprise calculating the two or more
base compositions from the two or more molecular masses of the two or
more amplification products. In some embodiments, the HPV bioagents are
selected from the group consisting of a HPV genus, a species thereof, a
sub-species thereof, and combinations thereof.

[0014] In some embodiments, the present invention provides methods of
identifying one or more strains of HPV in a sample, the method
comprising: (a) amplifying two or more segments of a nucleic acid from
the one or more HPV in the sample with first and second oligonucleotide
primer pairs to obtain two or more amplification products, wherein the
first primer pair is a broad range survey primer pair, and wherein the
second primer pair produces an amplicon that reveals species, sub-type,
strain, or genotype-specific information; (b) determining two or more
molecular masses and/or base compositions of the two or more
amplification products; and (c) comparing the two or more molecular
masses and/or the base compositions of the two or more amplification
products with known molecular masses and/or known base compositions of
amplification products of known HPV produced with the first and second
primer pairs to identify the HPV in the sample. In some embodiments, the
second primer pair amplifies a portion of a gene from HPV.

[0015] In certain embodiments, the second primer pair comprises forward
and reverse primers that are about 20 to 35 nucleobases in length, and
wherein the forward primer comprises at least 70% sequence identity with
a sequence selected from the group consisting of SEQ ID NOs: 1-8 and
17-43, and the reverse primer comprises at least 70% sequence identity
with a sequence selected from the group consisting of SEQ ID NOs: 9-16
and 44-70 to produce at least one amplification product. In further
embodiments, the obtaining the two or more molecular masses of the two or
more amplification products is via mass spectrometry. In some
embodiments, the methods comprise calculating the two or more base
compositions from the two or more molecular masses of the two or more
amplification products. In further embodiments, the HPV is selected from
the group consisting of: the family Papillomaviridae, the genus
Alphapapillomavirus, the genus Betapapillomavirus, the genus
Gammapapillomavirus, the genus Mupapillomavirus, and the genus
Nupapillomavirus.

[0016] In some embodiments, the second primer pair is selected from the
group of primer pair sequences consisting of: SEQ ID NOS: 1:9, 2:10,
3:11, 4:12, 5:13, 6:14, 7:15, 8:16, 17:44, 18:45, 19:46, 20:47, 21:48,
22:49, 23:50, 24:51, 25:52, 26:53, 27:54, 28:55, 29:56, 30:57, 31:58,
32:59, 33:60, 34:61, 35:62, 36:63, 37:64, 38:65, 39:66, 40:67, 41:68,
42:69, and 43:70. In other embodiments, the determining the two or more
molecular masses and/or base compositions is conducted without sequencing
the two or more amplification products. In certain embodiments, the HPV
in the sample cannot be identified using a single primer pair of the
first and second primer pairs. In other embodiments, the HPV in the
sample is identified by comparing three or more molecular masses and/or
base compositions of three or more amplification products with a database
of known molecular masses and/or known base compositions of amplification
products of known HPV produced with the first and second primer pairs,
and a third primer pair.

[0017] In further embodiments, members of the first and second primer
pairs hybridize to conserved regions of the nucleic acid that flank a
variable region. In some embodiments, the variable region varies between
at least two species of HPV. In particular embodiments, the variable
region uniquely varies between at least two (e.g., 3, 4, 5, 6, 7, 8, 9,
10, . . . , 20, etc.) genuses, species, sub-types, strains, or genotypes
of HPV.

[0018] In some embodiments, the present invention provides systems
comprising: (a) a mass spectrometer configured to detect one or more
molecular masses of amplicons produced using at least one purified
oligonucleotide primer pair that comprises forward and reverse primers
about 15 to 35 nucleobases in length, wherein the forward primer
comprises at least 70% (e.g., 70% . . . 75% . . . 90% . . . 95% . . .
100%) identity with a sequence selected from SEQ ID NOs: 1-8 and 17-43,
and wherein the reverse primer comprises at least 70% (e.g., 70% . . .
75% . . . 90% . . . 95% . . . 100%) identity with a sequence selected
from SEQ ID NOs: 9-16 and 44-70; and (b) a controller operably connected
to the mass spectrometer, the controller configured to correlate the
molecular masses of the amplicons with one or more species of HPV
identities. In certain embodiments, the second primer pair is selected
from the group of primer pair sequences consisting of: SEQ ID NOS: 1:9,
2:10, 3:11, 4:12, 5:13, 6:14, 7:15, 8:16, 17:44, 18:45, 19:46, 20:47,
21:48, 22:49, 23:50, 24:51, 25:52, 26:53, 27:54, 28:55, 29:56, 30:57,
31:58, 32:59, 33:60, 34:61, 35:62, 36:63, 37:64, 38:65, 39:66, 40:67,
41:68, 42:69, and 43:70. In other embodiments, the controller is
configured to determine base compositions of the amplicons from the
molecular masses of the amplicons, which base compositions correspond to
the one or more species of HPV. In particular embodiments, the controller
comprises or is operably connected to a database of known molecular
masses and/or known base compositions of amplicons of known species of
HPV produced with the primer pair.

[0019] In certain embodiments, the database comprises molecular mass
information for at least three different bioagents. In other embodiments,
the database comprises molecular mass information for at least 2 . . . 10
. . . 50 . . . 100 . . . 1000 . . . 10,000, or 100,000 different
bioagents. In particular embodiments, the molecular mass information
comprises base composition data. In some embodiments, the base
composition data comprises at least 10 . . . 50 . . . 100 . . . 500 . . .
1000 . . . 1000 . . . 10,000 . . . or 100,000 unique base compositions.
In other embodiments, the database comprises molecular mass information
for a bioagent from two or more genuses selected from the group
consisting of, but not limited to alphapapillomavirus,
betapapillomavirus, gammapapillomavirus. mupapillomavirus, and
nupapillomavirus. In some embodiments, the database comprises molecular
mass information for a bioagent from each of the genuses
alphapapillomavirus, betapapillomavirus, gammapapillomavirus,
mupapillomavirus, nupapillomavirus. In further embodiments, the database
comprises molecular mass information for a HPV bioagent. In further
embodiments, the database is stored on a local computer. In particular
embodiments, the database is accessed from a remote computer over a
network. In further embodiments, the molecular mass in the database is
associated with bioagent identity. In certain embodiments, the molecular
mass in the database is associated with bioagent geographic origin. In
particular embodiments, bioagent identification comprises interrogation
of the database with two or more different molecular masses (e.g., 2, 3,
4, 5, . . . 10 . . . 25 or more molecular masses) associated with the
bioagent.

[0023]FIG. 2 shows a process diagram illustrating one embodiment of the
primer pair validation process. Here select primers are shown meeting
test criteria. Criteria include but are not limited to, the ability to
amplify targeted HPV nucleic acid, the ability to exclude non-target
bioagents, the ability to not produce unexpected amplicons, the ability
to not dimerize, the ability to have analytical limits of detection of
≦100 genomic copies/reaction, and the ability to differentiate
amongst different target organisms.

[0026] It is to be understood that the terminology used herein is for the
purpose of describing particular embodiments only, and is not intended to
be limiting. Further, unless defined otherwise, all technical and
scientific terms used herein have the same meaning as commonly understood
by one of ordinary skill in the art to which this invention pertains. In
describing and claiming the present invention, the following terminology
and grammatical variants will be used in accordance with the definitions
set forth below.

[0027] As used herein, the term "about" means encompassing plus or minus
10%. For example, about 200 nucleotides refers to a range encompassing
between 180 and 220 nucleotides.

[0028] As used herein, the term "amplicon" or "bioagent identifying
amplicon" refers to a nucleic acid generated using the primer pairs
described herein. The amplicon is typically double stranded DNA; however,
it may be RNA and/or DNA:RNA. In some embodiments, the amplicon comprises
DNA complementary to HPV RNA, DNA, or cDNA. In some embodiments, the
amplicon comprises sequences of conserved regions/primer pairs and
intervening variable region. As discussed herein, primer pairs are
configured to generate amplicons from HPV nucleic acid. As such, the base
composition of any given amplicon may include the primer pair, the
complement of the primer pair, the conserved regions and the variable
region from the bioagent that was amplified to generate the amplicon. One
skilled in the art understands that the incorporation of the designed
primer pair sequences into an amplicon may replace the native sequences
at the primer binding site, and complement thereof. In certain
embodiments, after amplification of the target region using the primers
the resultant amplicons having the primer sequences are used to generate
the molecular mass data. Generally, the amplicon further comprises a
length that is compatible with mass spectrometry analysis. Bioagent
identifying amplicons generate base compositions that are preferably
unique to the identity of a bioagent (e.g., HPV).

[0030] The term "amplifying" or "amplification" in the context of nucleic
acids refers to the production of multiple copies of a polynucleotide, or
a portion of the polynucleotide, typically starting from a small amount
of the polynucleotide (e.g., a single polynucleotide molecule), where the
amplification products or amplicons are generally detectable.
Amplification of polynucleotides encompasses a variety of chemical and
enzymatic processes. The generation of multiple DNA copies from one or a
few copies of a target or template DNA molecule during a polymerase chain
reaction (PCR) or a ligase chain reaction (LCR) are forms of
amplification. Amplification is not limited to the strict duplication of
the starting molecule. For example, the generation of multiple cDNA
molecules from a limited amount of RNA in a sample using reverse
transcription (RT)-PCR is a form of amplification. Furthermore, the
generation of multiple RNA molecules from a single DNA molecule during
the process of transcription is also a form of amplification.

[0031] As used herein, "viral nucleic acid" includes, but is not limited
to, DNA, RNA, or DNA that has been obtained from viral RNA, such as, for
example, by performing a reverse transcription reaction. Viral RNA can
either be single-stranded (of positive or negative polarity) or
double-stranded.

[0032] As used herein, the term "base composition" refers to the number of
each residue comprised in an amplicon or other nucleic acid, without
consideration for the linear arrangement of these residues in the
strand(s) of the amplicon. The amplicon residues comprise, adenosine (A),
guanosine (G), cytidine, (C), (deoxy)thymidine (T), uracil (U), inosine
(1), nitroindoles such as 5-nitroindole or 3-nitropyrrole, dP or dK (Hill
F et al. Polymerase recognition of synthetic oligodeoxyribonucleotides
incorporating degenerate pyrimidine and purine bases. Proc Natl Acad Sci
USA. 1998 Apr. 14; 95(8):4258-63), an acyclic nucleoside analog
containing 5-nitroindazole (Van Aerschot et al., Nucleosides and
Nucleotides, 1995, 14, 1053-1056), the purine analog
1-(2-deoxy-beta-D-ribofuranosyl)-imidazole-4-carboxamide,
2,6-diaminopurine, 5-propynyluracil, 5-propynylcytosine, phenoxazines,
including G-clamp, 5-propynyl deoxy-cytidine, deoxy-thymidine
nucleotides, 5-propynylcytidine, 5-propynyluridine and mass tag modified
versions thereof, including 7-deaza-T-deoxyadenosine-5-triphosphate,
5-iodo-2'-deoxyuridine-5'-triphosphate,
5-bromo-2'-deoxyuridine-5'-triphosphate,
5-bromo-2'-deoxycytidine-5'-triphosphate,
5-iodo-2'-deoxycytidine-5'-triphosphate,
5-hydroxy-2'-deoxyuridine-5'-triphosphate,
4-thiothymidine-5'-triphosphate, 5-aza-2'-deoxyuridine-5'-triphosphate,
5-fluoro-2'-deoxyuridine-5'-triphosphate,
O6-methyl-2'-deoxyguanosine-5'-triphosphate,
N2-methyl-2'-deoxyguanosine-5'-triphosphate,
8-oxo-2'-deoxyguanosine-5'-triphosphate or thiothymidine-5'-triphosphate.
In some embodiments, the mass-modified nucleobase comprises 15N or
13C or both 15N and 13C. In some embodiments, the
non-natural nucleosides used herein include 5-propynyluracil,
5-propynylcytosine and inosine. Herein the base composition for an
unmodified DNA amplicon is notated as AwGxC.sub.yTz,
wherein w, x, y and z are each independently a whole number representing
the number of said nucleoside residues in an amplicon. Base compositions
for amplicons comprising modified nucleosides are similarly notated to
indicate the number of said natural and modified nucleosides in an
amplicon. Base compositions are calculated from a molecular mass
measurement of an amplicon, as described below. The calculated base
composition for any given amplicon is then compared to a database of base
compositions. A match between the calculated base composition and a
single database entry reveals the identity of the bioagent.

[0033] As used herein, a "base composition probability cloud" is a
representation of the diversity in base composition resulting from a
variation in sequence that occurs among different isolates of a given
species, family or genus. Base composition calculations for a plurality
of amplicons are mapped on a pseudo four-dimensional plot. Related
members in a family, genus or species typically cluster within this plot,
forming a base composition probability cloud.

[0034] As used herein, the term "base composition signature" refers to the
base composition generated by any one particular amplicon.

[0035] As used herein, a "bioagent" means any biological organism or
component thereof or a sample containing a biological organism or
component thereof, including microorganisms or infectious substances, or
any naturally occurring, bioengineered or synthesized component of any
such microorganism or infectious substance or any nucleic acid derived
from any such microorganism or infectious substance. Those of ordinary
skill in the art will understand fully what is meant by the term bioagent
given the instant disclosure. Still, a non-exhaustive list of bioagents
includes: cells, cell lines, human clinical samples, mammalian blood
samples, cell cultures, bacterial cells, viruses, viroids, fungi,
protists, parasites, rickettsiae, protozoa, animals, mammals or humans.
Samples may be alive, non-replicating or dead or in a vegetative state
(for example, vegetative bacteria or spores). Preferably, the bioagent is
an HPV such as Alphapapillomavirus or Gammapapillomavirus.

[0036] As used herein, a "bioagent division" is defined as group of
bioagents above the species level and includes but is not limited to,
orders, families, genus, classes, clades, genera or other such groupings
of bioagents above the species level.

[0037] As used herein, "broad range survey primers" are primers designed
to identify an unknown bioagent as a member of a particular biological
division (e.g., an order, family, class, Glade, or genus). However, in
some cases the broad range survey primers are also able to identify
unknown bioagents at the species or sub-species level. As used herein,
"division-wide primers" are primers designed to identify a bioagent at
the species level and "drill-down" primers are primers designed to
identify a bioagent at the sub-species level. As used herein, the
"sub-species" level of identification includes, but is not limited to,
strains, subtypes, variants, and isolates. Drill-down primers are not
always required for identification at the sub-species level because broad
range survey intelligent primers may, in some cases provide sufficient
identification resolution to accomplishing this identification objective.

[0038] As used herein, the terms "complementary" or "complementarity" are
used in reference to polynucleotides (i.e., a sequence of nucleotides)
related by the base-pairing rules. For example, the sequence
"5'-A-G-T-3'," is complementary to the sequence "3'-T-C-A-5'."
Complementarity may be "partial," in which only some of the nucleic
acids' bases are matched according to the base pairing rules. Or, there
may be "complete" or "total" complementarity between the nucleic acids.
The degree of complementarity between nucleic acid strands has
significant effects on the efficiency and strength of hybridization
between nucleic acid strands. This is of particular importance in
amplification reactions, as well as detection methods that depend upon
binding between nucleic acids.

[0039] The term "conserved region" in the context of nucleic acids refers
to a nucleobase sequence (e.g., a subsequence of a nucleic acid, etc.)
that is the same or similar in two or more different regions or segments
of a given nucleic acid molecule (e.g., an intramolecular conserved
region), or that is the same or similar in two or more different nucleic
acid molecules (e.g., an intermolecular conserved region). To illustrate,
a conserved region may be present in two or more different taxonomic
ranks (e.g., two or more different genera, two or more different species,
two or more different subspecies, and the like) or in two or more
different nucleic acid molecules from the same organism. To further
illustrate, in certain embodiments, nucleic acids comprising at least one
conserved region typically have between about 70%-100%, between about
80-100%, between about 90-100%, between about 95-100%, or between about
99-100% sequence identity in that conserved region. A conserved region
may also be selected or identified functionally as a region that permits
generation of amplicons via primer extension through hybridization of a
completely or partially complementary primer to the conserved region for
each of the target sequences to which conserved region is conserved.

[0040] The term "correlates" refers to establishing a relationship between
two or more things. In certain embodiments, for example, detected
molecular masses of one or more amplicons indicate the presence or
identity of a given bioagent in a sample. In some embodiments, base
compositions are calculated or otherwise determined from the detected
molecular masses of amplicons, which base compositions indicate the
presence or identity of a given bioagent in a sample.

[0041] As used herein, in some embodiments the term "database" is used to
refer to a collection of base composition molecular mass data. In other
embodiments the term "database" is used to refer to a collection of base
composition data. The base composition data in the database is indexed to
bioagents and to primer pairs. The base composition data reported in the
database comprises the number of each nucleoside in an amplicon that
would be generated for each bioagent using each primer. The database can
be populated by empirical data. In this aspect of populating the
database, a bioagent is selected and a primer pair is used to generate an
amplicon. The amplicon's molecular mass is determined using a mass
spectrometer and the base composition calculated therefrom without
sequencing i.e., without determining the linear sequence of nucleobases
comprising the amplicon. Note that base composition entries in the
database may be derived from sequencing data (i.e., known sequence
information), but the base composition of the amplicon to be identified
is determined without sequencing the amplicon. An entry in the database
is made to associate correlate the base composition with the bioagent and
the primer pair used. The database may also be populated using other
databases comprising bioagent information. For example, using the GenBank
database it is possible to perform electronic PCR using an electronic
representation of a primer pair. This in silico method may provide the
base composition for any or all selected bioagent(s) stored in the
GenBank database. The information may then be used to populate the base
composition database as described above. A base composition database can
be in silico, a written table, a reference book, a spreadsheet or any
form generally amenable to databases. Preferably, it is in silico on
computer readable media.

[0042] The term "detect", "detecting" or "detection" refers to an act of
determining the existence or presence of one or more targets (e.g.,
bioagent nucleic acids, amplicons, etc.) in a sample.

[0043] As used herein, the term "etiology" refers to the causes or
origins, of diseases or abnormal physiological conditions.

[0044] As used herein, the term "gene" refers to a nucleic acid (e.g.,
DNA) sequence that comprises coding sequences necessary for the
production of a polypeptide, precursor, or RNA (e.g., rRNA, tRNA). The
polypeptide can be encoded by a full length coding sequence or by any
portion of the coding sequence so long as the desired activity or
functional properties (e.g., enzymatic activity, ligand binding, signal
transduction, immunogenicity, etc.) of the full-length sequence or
fragment thereof are retained.

[0045] As used herein, the term "heterologous gene" refers to a gene that
is not in its natural environment. For example, a heterologous gene
includes a gene from one species introduced into another species. A
heterologous gene also includes a gene native to an organism that has
been altered in some way (e.g., mutated, added in multiple copies, linked
to non-native regulatory sequences, etc). Heterologous genes are
distinguished from endogenous genes in that the heterologous gene
sequences are typically joined to nucleic acid sequences that are not
found naturally associated with the gene sequences in the chromosome or
are associated with portions of the chromosome not found in nature (e.g.,
genes expressed in loci where the gene is not normally expressed).

[0046] The terms "homology," "homologous" and "sequence identity" refer to
a degree of identity. There may be partial homology or complete homology.
A partially homologous sequence is one that is less than 100% identical
to another sequence. Determination of sequence identity is described in
the following example: a primer 20 nucleobases in length which is
otherwise identical to another 20 nucleobase primer but having two
non-identical residues has 18 of 20 identical residues (18/20=0.9 or 90%
sequence identity). In another example, a primer 15 nucleobases in length
having all residues identical to a 15 nucleobase segment of a primer 20
nucleobases in length would have 15/20=0.75 or 75% sequence identity with
the 20 nucleobase primer. In context of the present invention, sequence
identity is meant to be properly determined when the query sequence and
the subject sequence are both described and aligned in the 5' to 3'
direction. Sequence alignment algorithms such as BLAST, will return
results in two different alignment orientations. In the Plus/Plus
orientation, both the query sequence and the subject sequence are aligned
in the 5' to 3' direction. On the other hand, in the Plus/Minus
orientation, the query sequence is in the 5' to 3' direction while the
subject sequence is in the 3' to 5' direction. It should be understood
that with respect to the primers of the present invention, sequence
identity is properly determined when the alignment is designated as
Plus/Plus. Sequence identity may also encompass alternate or "modified"
nucleobases that perform in a functionally similar manner to the regular
nucleobases adenine, thymine, guanine and cytosine with respect to
hybridization and primer extension in amplification reactions. In a
non-limiting example, if the 5-propynyl pyrimidines propyne C and/or
propyne T replace one or more C or T residues in one primer which is
otherwise identical to another primer in sequence and length, the two
primers will have 100% sequence identity with each other. In another
non-limiting example, Inosine (1) may be used as a replacement for G or T
and effectively hybridize to C, A or U (uracil). Thus, if inosine
replaces one or more C, A or U residues in one primer which is otherwise
identical to another primer in sequence and length, the two primers will
have 100% sequence identity with each other. Other such modified or
universal bases may exist which would perform in a functionally similar
manner for hybridization and amplification reactions and will be
understood to fall within this definition of sequence identity.

[0047] As used herein, "housekeeping gene" or "core viral gene" refers to
a gene encoding a protein or RNA involved in basic functions required for
survival and reproduction of a bioagent. Housekeeping genes include, but
are not limited to, genes encoding RNA or proteins involved in
translation, replication, recombination and repair, transcription,
nucleotide metabolism, amino acid metabolism, lipid metabolism, energy
generation, uptake, secretion and the like.

[0048] As used herein, the term "hybridization" or "hybridize" is used in
reference to the pairing of complementary nucleic acids. Hybridization
and the strength of hybridization (i.e., the strength of the association
between the nucleic acids) is influenced by such factors as the degree of
complementary between the nucleic acids, stringency of the conditions
involved, the melting temperature (Tm) of the formed hybrid, and the
G:C ratio within the nucleic acids. A single molecule that contains
pairing of complementary nucleic acids within its structure is said to be
"self-hybridized." An extensive guide to nucleic hybridization may be
found in Tijssen, Laboratory Techniques in Biochemistry and Molecular
Biology-Hybridization with Nucleic Acid Probes, part I, chapter 2,
"Overview of principles of hybridization and the strategy of nucleic acid
probe assays," Elsevier (1993), which is incorporated by reference.

[0049] As used herein, the term "primer" refers to an oligonucleotide,
whether occurring naturally as in a purified restriction digest or
produced synthetically, that is capable of acting as a point of
initiation of synthesis when placed under conditions in which synthesis
of a primer extension product that is complementary to a nucleic acid
strand is induced (e.g., in the presence of nucleotides and an inducing
agent such as a biocatalyst (e.g., a DNA polymerase or the like) and at a
suitable temperature and pH). The primer is typically single stranded for
maximum efficiency in amplification, but may alternatively be double
stranded. If double stranded, the primer is generally first treated to
separate its strands before being used to prepare extension products. In
some embodiments, the primer is an oligodeoxyribonucleotide. The primer
is sufficiently long to prime the synthesis of extension products in the
presence of the inducing agent. The exact lengths of the primers will
depend on many factors, including temperature, source of primer and the
use of the method.

[0050] As used herein, "intelligent primers" or "primers" or "primer
pairs," in some embodiments, are oligonucleotides that are designed to
bind to conserved sequence regions of one or more bioagent nucleic acids
to generate bioagent identifying amplicons. In some embodiments, the
bound primers flank an intervening variable region between the conserved
binding sequences. Upon amplification, the primer pairs yield amplicons
e.g., amplification products that provide base composition variability
between the two or more bioagents. The variability of the base
compositions allows for the identification of one or more individual
bioagents from, e.g., two or more bioagents based on the base composition
distinctions. In some embodiments, the primer pairs are also configured
to generate amplicons amenable to molecular mass analysis. Further, the
sequences of the primer members of the primer pairs are not necessarily
fully complementary to the conserved region of the reference bioagent.
For example, in some embodiments, the sequences are designed to be "best
fit" amongst a plurality of bioagents at these conserved binding
sequences. Therefore, the primer members of the primer pairs have
substantial complementarity with the conserved regions of the bioagents,
including the reference bioagent.

[0051] In some embodiments of the invention, the oligonucleotide primer
pairs described herein can be purified. As used herein, "purified
oligonucleotide primer pair," "purified primer pair," or "purified" means
an oligonucleotide primer pair that is chemically-synthesized to have a
specific sequence and a specific number of linked nucleosides. This term
is meant to explicitly exclude nucleotides that are generated at random
to yield a mixture of several compounds of the same length each with
randomly generated sequence. As used herein, the term "purified" or "to
purify" refers to the removal of one or more components (e.g.,
contaminants) from a sample.

[0052] As used herein, the term "molecular mass" refers to the mass of a
compound as determined using mass spectrometry, for example, ESI-MS.
Herein, the compound is preferably a nucleic acid. In some embodiments,
the nucleic acid is a double stranded nucleic acid (e.g., a double
stranded DNA nucleic acid). In some embodiments, the nucleic acid is an
amplicon. When the nucleic acid is double stranded the molecular mass is
determined for both strands. In one embodiment, the strands may be
separated before introduction into the mass spectrometer, or the strands
may be separated by the mass spectrometer (for example, electro-spray
ionization will separate the hybridized strands). The molecular mass of
each strand is measured by the mass spectrometer.

[0054] As used herein, the term "nucleobase" is synonymous with other
terms in use in the art including "nucleotide," "deoxynucleotide,"
"nucleotide residue," "deoxynucleotide residue," "nucleotide triphosphate
(NTP)," or deoxynucleotide triphosphate (dNTP). As is used herein, a
nucleobase includes natural and modified residues, as described herein.

[0055] An "oligonucleotide" refers to a nucleic acid that includes at
least two nucleic acid monomer units (e.g., nucleotides), typically more
than three monomer units, and more typically greater than ten monomer
units. The exact size of an oligonucleotide generally depends on various
factors, including the ultimate function or use of the oligonucleotide.
To further illustrate, oligonucleotides are typically less than 200
residues long (e.g., between 15 and 100), however, as used herein, the
term is also intended to encompass longer polynucleotide chains.
Oligonucleotides are often referred to by their length. For example a 24
residue oligonucleotide is referred to as a "24-mer". Typically, the
nucleoside monomers are linked by phosphodiester bonds or analogs
thereof, including phosphorothioate, phosphorodithioate,
phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,
phosphoranilidate, phosphoramidate, and the like, including associated
counterions, e.g., H.sup.+, NH4.sup.+, Na.sup.+, and the like, if
such counterions are present. Further, oligonucleotides are typically
single-stranded. Oligonucleotides are optionally prepared by any suitable
method, including, but not limited to, isolation of an existing or
natural sequence, DNA replication or amplification, reverse
transcription, cloning and restriction digestion of appropriate
sequences, or direct chemical synthesis by a method such as the
phosphotriester method of Narang et al. (1979) Meth Enzymol. 68: 90-99;
the phosphodiester method of Brown et al. (1979) Meth Enzymol. 68:
109-151; the diethylphosphoramidite method of Beaucage et al. (1981)
Tetrahedron Lett. 22: 1859-1862; the triester method of Matteucci et al.
(1981) J Am Chem. Soc. 103:3185-3191; automated synthesis methods; or the
solid support method of U.S. Pat. No. 4,458,066, entitled "PROCESS FOR
PREPARING POLYNUCLEOTIDES," issued Jul. 3, 1984 to Caruthers et al., or
other methods known to those skilled in the art. All of these references
are incorporated by reference.

[0056] As used herein a "sample" refers to anything capable of being
analyzed by the methods provided herein. In some embodiments, the sample
comprises or is suspected to comprise one or more nucleic acids capable
of analysis by the methods. Preferably, the samples comprise nucleic
acids (e.g., DNA, RNA, cDNAs, etc.) from one or more HPV. Samples can
include, for example, blood, saliva, urine, feces, anorectal swabs,
vaginal swabs, cervical swabs, and the like. In some embodiments, the
samples are "mixture" samples, which comprise nucleic acids from more
than one subject or individual. In some embodiments, the methods provided
herein comprise purifying the sample or purifying the nucleic acid(s)
from the sample. In some embodiments, the sample is purified nucleic
acid.

[0057] A "sequence" of a biopolymer refers to the order and identity of
monomer units (e.g., nucleotides, etc.) in the biopolymer. The sequence
(e.g., base sequence) of a nucleic acid is typically read in the 5' to 3'
direction.

[0058] As is used herein, the term "single primer pair identification"
means that one or more bioagents can be identified using a single primer
pair. A base composition signature for an amplicon may singly identify
one or more bioagents.

[0059] As used herein, a "sub-species characteristic" is a genetic
characteristic that provides the means to distinguish two members of the
same bioagent species. For example, one viral strain may be distinguished
from another viral strain of the same species by possessing a genetic
change (e.g., for example, a nucleotide deletion, addition or
substitution) in one of the viral genes, such as the RNA-dependent RNA
polymerase.

[0060] As used herein, in some embodiments the term "substantial
complementarity" means that a primer member of a primer pair comprises
between about 70%-100%, or between about 80-100%, or between about
90-100%, or between about 95-100%, or between about 99-100%
complementarity with the conserved binding sequence of a nucleic acid
from a given bioagent. Similarly, the primer pairs provided herein may
comprise between about 70%-100%, or between about 80-100%, or between
about 90-100%, or between about 95-100% identity, or between about
99-100% sequence identity with the primer pairs disclosed in Tables 1 and
2. These ranges of complementarity and identity are inclusive of all
whole or partial numbers embraced within the recited range numbers. For
example, and not limitation, 75.667%, 82%, 91.2435% and 97%
complementarity or sequence identity are all numbers that fall within the
above recited range of 70% to 100%, therefore forming a part of this
description. In some embodiments, any oligonucleotide primer pair may
have one or both primers with less then 70% sequence homology with a
corresponding member of any of the primer pairs of Tables 1 and 2 if the
primer pair has the capability of producing an amplification product
corresponding to the desired HPV identifying amplicon.

[0061] A "system" in the context of analytical instrumentation refers a
group of objects and/or devices that form a network for performing a
desired objective.

[0062] As used herein, "triangulation identification" means the use of
more than one primer pair to generate a corresponding amplicon for
identification of a bioagent. The more than one primer pair can be used
in individual wells or vessels or in a multiplex PCR assay.
Alternatively, PCR reactions may be carried out in single wells or
vessels comprising a different primer pair in each well or vessel.
Following amplification the amplicons are pooled into a single well or
container which is then subjected to molecular mass analysis. The
combination of pooled amplicons can be chosen such that the expected
ranges of molecular masses of individual amplicons are not overlapping
and thus will not complicate identification of signals. Triangulation is
a process of elimination, wherein a first primer pair identifies that an
unknown bioagent may be one of a group of bioagents. Subsequent primer
pairs are used in triangulation identification to further refine the
identity of the bioagent amongst the subset of possibilities generated
with the earlier primer pair. Triangulation identification is complete
when the identity of the bioagent is determined. The triangulation
identification process may also be used to reduce false negative and
false positive signals, and enable reconstruction of the origin of hybrid
or otherwise engineered bioagents. For example, identification of the
three part toxin genes typical of B. anthracis (Bowen et al., J Appl
Microbiol., 1999, 87, 270-278) in the absence of the expected
compositions from the B. anthracis genome would suggest a genetic
engineering event.

[0063] As used herein, the term "unknown bioagent" can mean, for example:
(i) a bioagent whose existence is not known (for example, the SARS
coronavirus was unknown prior to April 2003) and/or (ii) a bioagent whose
existence is known (such as the well known bacterial species
Staphylococcus aureus for example) but which is not known to be in a
sample to be analyzed. For example, if the method for identification of
coronaviruses disclosed in commonly owned U.S. patent Ser. No. 10/829,826
(incorporated herein by reference in its entirety) was to be employed
prior to April 2003 to identify the SARS coronavirus in a clinical
sample, both meanings of "unknown" bioagent are applicable since the SARS
coronavirus was unknown to science prior to April, 2003 and since it was
not known what bioagent (in this case a coronavirus) was present in the
sample. On the other hand, if the method of U.S. patent Ser. No.
10/829,826 was to be employed subsequent to April 2003 to identify the
SARS coronavirus in a clinical sample, the second meaning (ii) of
"unknown" bioagent would apply because the SARS coronavirus became known
to science subsequent to April 2003 because it was not known what
bioagent was present in the sample.

[0064] As used herein, the term "variable region" is used to describe a
region that falls between any one primer pair described herein. The
region possesses distinct base compositions between at least two
bioagents, such that at least one bioagent can be identified at, for
example, the family, genus, species or sub-species level. The degree of
variability between the at least two bioagents need only be sufficient to
allow for identification using mass spectrometry analysis, as described
herein.

[0065] As used herein, a "wobble base" is a variation in a codon found at
the third nucleotide position of a DNA triplet. Variations in conserved
regions of sequence are often found at the third nucleotide position due
to redundancy in the amino acid code.

[0066] Provided herein are methods, compositions, kits, and related
systems for the detection and identification of bioagents (e.g., species
of HPV) using bioagent identifying amplicons. In some embodiments,
primers are selected to hybridize to conserved sequence regions of
nucleic acids derived from a bioagent and which flank variable sequence
regions to yield a bioagent identifying amplicon which can be amplified
and which is amenable to molecular mass determination. In some
embodiments, the molecular mass is converted to a base composition, which
indicates the number of each nucleotide in the amplicon. Systems
employing software and hardware useful in converting molecular mass data
into base composition information are available from, for example, Ibis
Biosciences, Inc. (Carlsbad, Calif.), for example the Ibis T5000
Biosensor System, and are described in U.S. patent application Ser. No.
10/754,415, filed Jan. 9, 2004, incorporated by reference herein in its
entirety. In some embodiments, the molecular mass or corresponding base
composition of one or more different amplicons is queried against a
database of molecular masses or base compositions indexed to bioagents
and to the primer pair used to generate the amplicon. A match of the
measured base composition to a database entry base composition associates
the sample bioagent to an indexed bioagent in the database. Thus, the
identity of the unknown bioagent is determined. No prior knowledge of the
unknown bioagent is necessary to make an identification. In some
instances, the measured base composition associates with more than one
database entry base composition. Thus, a second/subsequent primer pair is
generally used to generate an amplicon, and its measured base composition
is similarly compared to the database to determine its identity in
triangulation identification. Furthermore, the methods and other aspects
of the invention can be applied to rapid parallel multiplex analyses, the
results of which can be employed in a triangulation identification
strategy. Thus, in some embodiments, the present invention provides rapid
throughput and does not require nucleic acid sequencing or knowledge of
the linear sequences of nucleobases of the amplified target sequence for
bioagent detection and identification.

[0069] In certain embodiments, bioagent identifying amplicons amenable to
molecular mass determination produced by the primers described herein are
either of a length, size or mass compatible with a particular mode of
molecular mass determination, or compatible with a means of providing a
fragmentation pattern in order to obtain fragments of a length compatible
with a particular mode of molecular mass determination. Such means of
providing a fragmentation pattern of an amplicon include, but are not
limited to, cleavage with restriction enzymes or cleavage primers,
sonication or other means of fragmentation. Thus, in some embodiments,
bioagent identifying amplicons are larger than 200 nucleobases and are
amenable to molecular mass determination following restriction digestion.
Methods of using restriction enzymes and cleavage primers are well known
to those with ordinary skill in the art.

[0071] One embodiment of a process flow diagram used for primer selection
and validation process is depicted in FIGS. 1 and 2. For each group of
organisms, candidate target sequences are identified (200) from which
nucleotide sequence alignments are created (210) and analyzed (220).
Primers are then configured by selecting priming regions (230) to
facilitate the selection of candidate primer pairs (240). The primer pair
sequence is typically a "best fit" amongst the aligned sequences, such
that the primer pair sequence may or may not be fully complementary to
the hybridization region on any one of the bioagents in the alignment.
Thus, best fit primer pair sequences are those with sufficient
complementarity with two or more bioagents to hybridize with the two or
more bioagents and generate an amplicon. The primer pairs are then
subjected to in silico analysis by electronic PCR (ePCR) (300) wherein
bioagent identifying amplicons are obtained from sequence databases such
as GenBank or other sequence collections (310) and tested for specificity
in silico (320). Bioagent identifying amplicons obtained from ePCR of
GenBank sequences (310) may also be analyzed by a probability model which
predicts the capability of a given amplicon to identify unknown
bioagents. Preferably, the base compositions of amplicons with favorable
probability scores are then stored in a base composition database (325).
Alternatively, base compositions of the bioagent identifying amplicons
obtained from the primers and GenBank sequences are directly entered into
the base composition database (330). Candidate primer pairs (240) are
validated by in vitro amplification by a method such as PCR analysis
(400) of nucleic acid from a collection of organisms (410). Amplicons
thus obtained are analyzed to confirm the sensitivity, specificity and
reproducibility of the primers used to obtain the amplicons (420).

[0072] Synthesis of primers is well known and routine in the art. The
primers may be conveniently and routinely made through the well-known
technique of solid phase synthesis. Equipment for such synthesis is sold
by several vendors including, for example, Applied Biosystems (Foster
City, Calif.). Any other means for such synthesis known in the art may
additionally or alternatively be employed.

[0073] The primers typically are employed as compositions for, use in
methods for identification of bioagents as follows: a primer pair
composition is contacted with nucleic acid of an unknown isolate
suspected of comprising HPV. The nucleic acid is then amplified by a
nucleic acid amplification technique, such as PCR for example, to obtain
an amplicon that represents a bioagent identifying amplicon. The
molecular mass of the strands of the double-stranded amplicon is
determined by a molecular mass measurement technique such as mass
spectrometry, for example. Preferably the two strands of the
double-stranded amplicon are separated during the ionization process;
however, they may be separated prior to mass spectrometry measurement. In
some embodiments, the mass spectrometer is electrospray Fourier transform
ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) or electrospray
time of flight mass spectrometry (ESI-TOF-MS). A list of possible base
compositions may be generated for the molecular mass value obtained for
each strand, and the choice of the base composition from the list is
facilitated by matching the base composition of one strand with a
complementary base composition of the other strand. A measured molecular
mass or base composition calculated therefrom is then compared with a
database of molecular masses or base compositions indexed to primer pairs
and to known bioagents. A match between the measured molecular mass or
base composition of the amplicon and the database molecular mass or base
composition for that indexed primer pair correlates the measured
molecular mass or base composition with an indexed bioagent, thus
identifying the unknown bioagent (e.g. the species of HPV). In some
embodiments, the primer pair used is at least one of the primer pairs of
Tables 1 and 2. In some embodiments, the method is repeated using a
different primer pair to resolve possible ambiguities in the
identification process or to improve the confidence level for the
identification assignment (triangulation identification). In some
embodiments, for example, where the unknown is a novel, previously
uncharacterized organism, the molecular mass or base composition from an
amplicon generated from the unknown is matched with one or more best
match molecular masses or base compositions from a database to predict a
family, genus, species, sub-type, etc. of the unknown. Such information
may assist further characterization of the unknown or provide a physician
treating a patient infected by the unknown with a therapeutic agent best
calculated to treat the patient.

[0074] In certain embodiments, HPV is detected with the systems and
methods of the present invention in combination with other bioagents,
including viruses, bacteria, fungi, or other bioagents. In particular
embodiments, a panel is employed that includes HPV and other related or
un-related bioagents. Such panels may be specific for a particular type
of bioagent, or specific for a specific type of test (e.g., for testing
the safety of blood, one may include commonly present viral pathogens
such as HCV, HIV, and bacteria that can be contracted via a blood
transfusion).

[0075] In some embodiments, a bioagent identifying amplicon may be
produced using only a single primer (either the forward or reverse primer
of any given primer pair), provided an appropriate amplification method
is chosen, such as, for example, low stringency single primer PCR
(LSSP-PCR).

[0076] In some embodiments, the oligonucleotide primers are broad range
survey primers which hybridize to conserved regions of nucleic acid. The
broad range primer may identify the unknown bioagent depending on which
bioagent is in the sample. In other cases, the molecular mass or base
composition of an amplicon does not provide sufficient resolution to
identify the unknown bioagent as any one bioagent at or below the species
level. These cases generally benefit from further analysis of one or more
amplicons generated from at least one additional broad range survey
primer pair, or from at least one additional division-wide primer pair,
or from at least one additional drill-down primer pair. Identification of
sub-species characteristics may be required, for example, to determine a
clinical treatment of patient, or in rapidly responding to an outbreak of
a new species, sub-type, etc. of pathogen to prevent an epidemic or
pandemic.

[0077] One with ordinary skill in the art of design of amplification
primers will recognize that a given primer need not hybridize with 100%
complementarity in order to effectively prime the synthesis of a
complementary nucleic acid strand in an amplification reaction. Primer
pair sequences may be a "best fit" amongst the aligned bioagent
sequences, thus they need not be fully complementary to the hybridization
region of any one of the bioagents in the alignment. Moreover, a primer
may hybridize over one or more segments such that intervening or adjacent
segments are not involved in the hybridization event (e.g., for example,
a loop structure or a hairpin structure). The primers may comprise at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at
least 95% or at least 99% sequence identity with any of the primers
listed in Tables 1 and 2. Thus, in some embodiments, an extent of
variation of 70% to 100%, or any range falling within, of the sequence
identity is possible relative to the specific primer sequences disclosed
herein. To illustrate, determination of sequence identity is described in
the following example: a primer 20 nucleobases in length which is
identical to another 20 nucleobase primer having two non-identical
residues has 18 of 20 identical residues (18/20=0.9 or 90% sequence
identity). In another example, a primer 15 nucleobases in length having
all residues identical to a 15 nucleobase segment of primer 20
nucleobases in length would have 15/20=0.75 or 75% sequence identity with
the 20 nucleobase primer. Percent identity need not be a whole number,
for example when a 28 consecutive nucleobase primer is completely
identical to a 31 consecutive nucleobase primer (28/31=0.9032 or 90.3%
identical).

[0078] Percent homology, sequence identity or complementarity, can be
determined by, for example, the Gap program (Wisconsin Sequence Analysis
Package, Version 8 for Unix, Genetics Computer Group, University Research
Park, Madison Wis.), using default settings, which uses the algorithm of
Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489). In some
embodiments, complementarity of primers with respect to the conserved
priming regions of viral nucleic acid, is between about 70% and about
80%. In other embodiments, homology, sequence identity or
complementarity, is between about 80% and about 90%. In yet other
embodiments, homology, sequence identity or complementarity, is at least
90%, at least 92%, at least 94%, at least 95%, at least 96%, at least
97%, at least 98%, at least 99% or is 100%.

[0079] In some embodiments, the primers described herein comprise at least
70%, at least 75%, at least 80%, at least 85%, at least 90%, at least
92%, at least 94%, at least 95%, at least 96%, at least 98%, or at least
99%, or 100% (or any range falling within) sequence identity with the
primer sequences specifically disclosed herein.

[0081] In some embodiments, any given primer comprises a modification
comprising the addition of a non-templated T residue to the 5' end of the
primer (i.e., the added T residue does not necessarily hybridize to the
nucleic acid being amplified). The addition of a non-templated T residue
has an effect of minimizing the addition of non-templated A residues as a
result of the non-specific enzyme activity of, e.g., Taq DNA polymerase
(Magnuson et al., Biotechniques, 1996, 21, 700-709), an occurrence which
may lead to ambiguous results arising from molecular mass analysis.

[0082] Primers may contain one or more universal bases. Because any
variation (due to codon wobble in the third position) in the conserved
regions among species is likely to occur in the third position of a DNA
(or RNA) triplet, oligonucleotide primers can be designed such that the
nucleotide corresponding to this position is a base which can bind to
more than one nucleotide, referred to herein as a "universal nucleobase."
For example, under this "wobble" base pairing, inosine (1) binds to U, C
or A; guanine (G) binds to U or C, and uridine (U) binds to U or C. Other
examples of universal nucleobases include nitroindoles such as
5-nitroindole or 3-nitropyrrole (Loakes et al., Nucleosides and
Nucleotides, 1995, 14, 1001-1003), the degenerate nucleotides dP or dK,
an acyclic nucleoside analog containing 5-nitroindazole (Van Aerschot et
al., Nucleosides and Nucleotides., 1995, 14, 1053-1056) or the purine
analog 1-(2-deoxy-beta-D-ribofuranosyl)-imidazole-4-carboxamide (Sala et
al., Nucl. Acids Res., 1996, 24, 3302-3306).

[0083] In some embodiments, to compensate for weaker binding by the wobble
base, oligonucleotide primers are configured such that the first and
second positions of each triplet are occupied by nucleotide analogs which
bind with greater affinity than the unmodified nucleotide. Examples of
these analogs include, but are not limited to, 2,6-diaminopurine which
binds to thymine, 5-propynyluracil which binds to adenine and
5-propynylcytosine and phenoxazines, including G-clamp, which binds to G.
Propynylated pyrimidines are described in U.S. Pat. Nos. 5,645,985,
5,830,653 and 5,484,908, each of which is commonly owned and incorporated
herein by reference in its entirety. Propynylated primers are described
in U.S Pre-Grant Publication No. 2003-0170682; also commonly owned and
incorporated herein by reference in its entirety. Phenoxazines are
described in U.S. Pat. Nos. 5,502,177, 5,763,588, and 6,005,096, each of
which is incorporated herein by reference in its entirety. G-clamps are
described in U.S. Pat. Nos. 6,007,992 and 6,028,183, each of which is
incorporated herein by reference in its entirety.

[0084] In some embodiments, non-template primer tags are used to increase
the melting temperature (Tm) of a primer-template duplex in order to
improve amplification efficiency. A non-template tag is at least three
consecutive A or T nucleotide residues on a primer which are not
complementary to the template. In any given non-template tag, A can be
replaced by C or G and T can also be replaced by C or G. Although
Watson-Crick hybridization is not expected to occur for a non-template
tag relative to the template, the extra hydrogen bond in a G-C pair
relative to an A-T pair confers increased stability of the
primer-template duplex and improves amplification efficiency for
subsequent cycles of amplification when the primers hybridize to strands
synthesized in previous cycles.

[0085] In other embodiments, propynylated tags may be used in a manner
similar to that of the non-template tag, wherein two or more
5-propynylcytidine or 5-propynyluridine residues replace template
matching residues on a primer. In other embodiments, a primer contains a
modified internucleoside linkage such as a phosphorothioate linkage, for
example.

[0086] In some embodiments, the primers contain mass-modifying tags.
Reducing the total number of possible base compositions of a nucleic acid
of specific molecular weight provides a means of avoiding a possible
source of ambiguity in the determination of base composition of
amplicons. Addition of mass-modifying tags to certain nucleobases of a
given primer will result in simplification of de novo determination of
base composition of a given bioagent identifying amplicon from its
molecular mass.

[0087] In some embodiments, the mass modified nucleobase comprises one or
more of the following: for example,
7-deaza-2'-deoxyadenosine-5-triphosphate,
5-iodo-2'-deoxyuridine-5'-triphosphate,
5-bromo-2'-deoxyuridine-5'-triphosphate,
5-bromo-2'-deoxycytidine-5'-triphosphate,
5-iodo-2'-deoxycytidine-5'-triphosphate,
5-hydroxy-2'-deoxyuridine-5'-triphosphate,
4-thiothymidine-5'-triphosphate, 5-aza-2'-deoxyuridine-5'-triphosphate,
5-fluoro-2'-deoxyuridine-5'-triphosphate,
O6-methyl-2'-deoxyguanosine-5'-triphosphate,
N2-methyl-2'-deoxyguanosine-5'-triphosphate,
8-oxo-2'-deoxyguanosine-5'-triphosphate or thiothymidine-5'-triphosphate.
In some embodiments, the mass-modified nucleobase comprises 15N or
13C or both 13N and 13C.

[0088] In some embodiments, the molecular mass of a given bioagent (e.g.,
a species of HPV) identifying amplicon is determined by mass
spectrometry. Mass spectrometry is intrinsically a parallel detection
scheme without the need for radioactive or fluorescent labels, because an
amplicon is identified by its molecular mass. The current state of the
art in mass spectrometry is such that less than femtomole quantities of
material can be analyzed to provide information about the molecular
contents of the sample. An accurate assessment of the molecular mass of
the material can be quickly obtained, irrespective of whether the
molecular weight of the sample is several hundred, or in excess of one
hundred thousand atomic mass units (amu) or Daltons.

[0089] In some embodiments, intact molecular ions are generated from
amplicons using one of a variety of ionization techniques to convert the
sample to the gas phase. These ionization methods include, but are not
limited to, electrospray ionization (ESI), matrix-assisted laser
desorption ionization (MALDI) and fast atom bombardment (FAB). Upon
ionization, several peaks are observed from one sample due to the
formation of ions with different charges. Averaging the multiple readings
of molecular mass obtained from a single mass spectrum affords an
estimate of molecular mass of the bioagent identifying amplicon.
Electrospray ionization mass spectrometry (ESI-MS) is particularly useful
for very high molecular weight polymers such as proteins and nucleic
acids having molecular weights greater than 10 kDa, since it yields a
distribution of multiply-charged molecules of the sample without causing
a significant amount of fragmentation.

[0091] In some embodiments, assignment of previously unobserved base
compositions (also known as "true unknown base compositions") to a given
phylogeny can be accomplished via the use of pattern classifier model
algorithms. Base compositions, like sequences, may vary slightly from
strain to strain within species, for example. In some embodiments, the
pattern classifier model is the mutational probability model. In other
embodiments, the pattern classifier is the polytope model. A polytope
model is the mutational probability model that incorporates both the
restrictions among strains and position dependence of a given nucleobase
within a triplet. In certain embodiments, a polytope pattern classifier
is used to classify a test or unknown organism according to its amplicon
base composition.

[0092] In some embodiments, it is possible to manage this diversity by
building "base composition probability clouds" around the composition
constraints for each species. A "pseudo four-dimensional plot" may be
used to visualize the concept of base composition probability clouds.
Optimal primer design typically involves an optimal choice of bioagent
identifying amplicons and maximizes the separation between the base
composition signatures of individual bioagents. Areas where clouds
overlap generally indicate regions that may result in a
misclassification, a problem which is overcome by a triangulation
identification process using bioagent identifying amplicons not affected
by overlap of base composition probability clouds.

[0093] In some embodiments, base composition probability clouds provide
the means for screening potential primer pairs in order to avoid
potential misclassifications of base compositions. In other embodiments,
base composition probability clouds provide the means for predicting the
identity of an unknown bioagent whose assigned base composition has not
been previously observed and/or indexed in a bioagent identifying
amplicon base composition database due to evolutionary transitions in its
nucleic acid sequence. Thus, in contrast to probe-based techniques, mass
spectrometry determination of base composition does not require prior
knowledge of the composition or sequence in order to make the
measurement.

[0094] Provided herein is bioagent classifying information at a level
sufficient to identify a given bioagent. Furthermore, the process of
determining a previously unknown base composition for a given bioagent
(for example, in a case where sequence information is unavailable) has
utility by providing additional bioagent indexing information with which
to populate base composition databases. The process of future bioagent
identification is thus improved as additional base composition signature
indexes become available in base composition databases.

[0095] In some embodiments, the identity and quantity of an unknown
bioagent may be determined using the process illustrated in FIG. 3.
Primers (500) and a known quantity of a calibration polynucleotide (505)
are added to a sample containing nucleic acid of an unknown bioagent. The
total nucleic acid in the sample is then subjected to an amplification
reaction (510) to obtain amplicons. The molecular masses of amplicons are
determined (515) from which are obtained molecular mass and abundance
data. The molecular mass of the bioagent identifying amplicon (520)
provides for its identification (525) and the molecular mass of the
calibration amplicon obtained from the calibration polynucleotide (530)
provides for its quantification (535). The abundance data of the bioagent
identifying amplicon is recorded (540) and the abundance data for the
calibration data is recorded (545), both of which are used in a
calculation (550) which determines the quantity of unknown bioagent in
the sample.

[0096] In certain embodiments, a sample comprising an unknown bioagent is
contacted with a primer pair which amplifies the nucleic acid from the
bioagent, and a known quantity of a polynucleotide that comprises a
calibration sequence. The amplification reaction then produces two
amplicons: a bioagent identifying amplicon and a calibration amplicon.
The bioagent identifying amplicon and the calibration amplicon are
distinguishable by molecular mass while being amplified at essentially
the same rate.

[0097] Effecting differential molecular masses can be accomplished by
choosing as a calibration sequence, a representative bioagent identifying
amplicon (from a specific species of bioagent) and performing, for
example, a 2-8 nucleobase deletion or insertion within the variable
region between the two priming sites. The amplified sample containing the
bioagent identifying amplicon and the calibration amplicon is then
subjected to molecular mass analysis by mass spectrometry, for example.
The resulting molecular mass analysis of the nucleic acid of the bioagent
and of the calibration sequence provides molecular mass data and
abundance data for the nucleic acid of the bioagent and of the
calibration sequence. The molecular mass data obtained for the nucleic
acid of the bioagent enables identification of the unknown bioagent by
base composition analysis. The abundance data enables calculation of the
quantity of the bioagent, based on the knowledge of the quantity of
calibration polynucleotide contacted with the sample.

[0098] In some embodiments, construction of a standard curve in which the
amount of calibration or calibrant polynucleotide spiked into the sample
is varied provides additional resolution and improved confidence for the
determination of the quantity of bioagent in the sample. Alternatively,
the calibration polynucleotide can be amplified in its own reaction
vessel or vessels under the same conditions as the bioagent. A standard
curve may be prepared there from, and the relative abundance of the
bioagent determined by methods such as linear regression. In some
embodiments, multiplex amplification is performed where multiple bioagent
identifying amplicons are amplified with multiple primer pairs which also
amplify the corresponding standard calibration sequences. In this or
other embodiments, the standard calibration sequences are optionally
included within a single construct (preferably a vector) which functions
as the calibration polynucleotide.

[0099] In some embodiments, the calibrant polynucleotide is used as an
internal positive control to confirm that amplification conditions and
subsequent analysis steps are successful in producing a measurable
amplicon. Even in the absence of copies of the genome of a bioagent, the
calibration polynucleotide gives rise to a calibration amplicon. Failure
to produce a measurable calibration amplicon indicates a failure of
amplification or subsequent analysis step such as amplicon purification
or molecular mass determination. Reaching a conclusion that such failures
have occurred is, in itself, a useful event. In some embodiments, the
calibration sequence is comprised of DNA. In some embodiments, the
calibration sequence is comprised of RNA.

[0100] In some embodiments, a calibration sequence is inserted into a
vector which then functions as the calibration polynucleotide. In some
embodiments, more than one calibration sequence is inserted into the
vector that functions as the calibration polynucleotide. Such a
calibration polynucleotide is herein termed a "combination calibration
polynucleotide." It should be recognized that the calibration method
should not be limited to the embodiments described herein. The
calibration method can be applied for determination of the quantity of
any bioagent identifying amplicon when an appropriate standard calibrant
polynucleotide sequence is designed and used.

[0101] In certain embodiments, primer pairs are configured to produce
bioagent identifying amplicons within more conserved regions of an HPV,
while others produce bioagent identifying amplicons within regions that
are may evolve more quickly. Primer pairs that characterize amplicons in
a conserved region with low probability that the region will evolve past
the point of primer recognition are useful, e.g., as a broad range
survey-type primer. Primer pairs that characterize an amplicon
corresponding to an evolving genomic region are useful, e.g., for
distinguishing emerging bioagent strain variants.

[0102] The primer pairs described herein provide reagents, e.g., for
identifying diseases caused by emerging types of HPV. Base composition
analysis eliminates the need for prior knowledge of bioagent sequence to
generate hybridization probes. Thus, in another embodiment, there is
provided a method for determining the etiology of a particular stain when
the process of identification of is carried out in a clinical setting,
and even when a new strain is involved. This is possible because the
methods may not be confounded by naturally occurring evolutionary
variations.

[0103] Another embodiment provides a means of tracking the spread of any
species or strain of HPV when a plurality of samples obtained from
different geographical locations are analyzed by methods described above
in an epidemiological setting. For example, a plurality of samples from a
plurality of different locations may be analyzed with primers which
produce bioagent identifying amplicons, a subset of which identifies a
specific strain. The corresponding locations of the members of the
strain-containing subset indicate the spread of the specific strain to
the corresponding locations.

[0104] Also provided are kits for carrying out the methods described
herein. In some embodiments, the kit may comprise a sufficient quantity
of one or more primer pairs to perform an amplification reaction on a
target polynucleotide from a bioagent to form a bioagent identifying
amplicon. In some embodiments, the kit may comprise from one to twenty
primer pairs, from one to ten primer pairs, from one to eight pairs, from
one to five primer pairs, from one to three primer pairs, or from one to
two primer pairs. In some embodiments, the kit may comprise one or more
primer pairs recited in Tables 1 and 2. In certain embodiments, kits
include all of the primer pairs recited in Table 1, or Table 2, or Tables
1 and 2.

[0105] In some embodiments, the kit may also comprise a sufficient
quantity of reverse transcriptase, a DNA polymerase, suitable nucleoside
triphosphates (including any of those described above), a DNA ligase,
and/or reaction buffer, or any combination thereof, for the amplification
processes described above. A kit may further include instructions
pertinent for the particular embodiment of the kit, such instructions
describing the primer pairs and amplification conditions for operation of
the method. In some embodiments, the kit further comprises instructions
for analysis, interpretation and dissemination of data acquired by the
kit. In other embodiments, instructions for the operation, analysis,
interpretation and dissemination of the data of the kit are provided on
computer readable media. A kit may also comprise amplification reaction
containers such as microcentrifuge tubes, microtiter plates, and the
like. A kit may also comprise reagents or other materials for isolating
bioagent nucleic acid or bioagent identifying amplicons from
amplification reactions, including, for example, detergents, solvents, or
ion exchange resins which may be linked to magnetic beads. A kit may also
comprise a table of measured or calculated molecular masses and/or base
compositions of bioagents using the primer pairs of the kit.

[0106] The invention also provides systems that can be used to perform
various assays relating to HPV detection or identification. In certain
embodiments, systems include mass spectrometers configured to detect
molecular masses of amplicons produced using purified oligonucleotide
primer pairs described herein. Other detectors that are optionally
adapted for use in the systems of the invention are described further
below. In some embodiments, systems also include controllers operably
connected to mass spectrometers and/or other system components. In some
of these embodiments, controllers are configured to correlate the
molecular masses of the amplicons with bioagents to effect detection or
identification. In some embodiments, controllers are configured to
determine base compositions of the amplicons from the molecular masses of
the amplicons. As described herein, the base compositions generally
correspond to the HPV species identities. In certain embodiments,
controllers include, or are operably connected to, databases of known
molecular masses and/or known base compositions of amplicons of known
species of HPV produced with the primer pairs described herein.
Controllers are described further below.

[0107] In some embodiments, systems include one or more of the primer
pairs described herein (e.g., in Tables 1 and 2). In certain embodiments,
the oligonucleotides are arrayed on solid supports, whereas in others,
they are provided in one or more containers, e.g., for assays performed
in solution. In certain embodiments, the systems also include at least
one detector or detection component (e.g., a spectrometer) that is
configured to detect detectable signals produced in the container or on
the support. In addition, the systems also optionally include at least
one thermal modulator (e.g., a thermal cycling device) operably connected
to the containers or solid supports to modulate temperature in the
containers or on the solid supports, and/or at least one fluid transfer
component (e.g., an automated pipettor) that transfers fluid to and/or
from the containers or solid supports, e.g., for performing one or more
assays (e.g., nucleic acid amplification, real-time amplicon detection,
etc.) in the containers or on the solid supports.

[0109] As mentioned above, the systems of the invention also typically
include controllers that are operably connected to one or more components
(e.g., detectors, databases, thermal modulators, fluid transfer
components, robotic material handling devices, and the like) of the given
system to control operation of the components. More specifically,
controllers are generally included either as separate or integral system
components that are utilized, e.g., to receive data from detectors (e.g.,
molecular masses, etc.), to effect and/or regulate temperature in the
containers, or to effect and/or regulate fluid flow to or from selected
containers. Controllers and/or other system components are optionally
coupled to an appropriately programmed processor, computer, digital
device, information appliance, or other logic device (e.g., including an
analog to digital or digital to analog converter as needed), which
functions to instruct the operation of these instruments in accordance
with preprogrammed or user input instructions, receive data and
information from these instruments, and interpret, manipulate and report
this information to the user. Suitable controllers are generally known in
the art and are available from various commercial sources.

[0110] Any controller or computer optionally includes a monitor, which is
often a cathode ray tube ("CRT") display, a flat panel display (e.g.,
active matrix liquid crystal display or liquid crystal display), or
others. Computer circuitry is often placed in a box, which includes
numerous integrated circuit chips, such as a microprocessor, memory,
interface circuits, and others. The box also optionally includes a hard
disk drive, a floppy disk drive, a high capacity removable drive such as
a writeable CD-ROM, and other common peripheral elements. Inputting
devices such as a keyboard or mouse optionally provide for input from a
user. These components are illustrated further below.

[0111] The computer typically includes appropriate software for receiving
user instructions, either in the form of user input into a set of
parameter fields, e.g., in a graphic user interface (GUI), or in the form
of preprogrammed instructions, e.g., preprogrammed for a variety of
different specific operations. The software then converts these
instructions to appropriate language for instructing the operation of one
or more controllers to carry out the desired operation. The computer then
receives the data from, e.g., sensors/detectors included within the
system, and interprets the data, either provides it in a user understood
format, or uses that data to initiate further controller instructions, in
accordance with the programming.

[0112]FIG. 4 is a schematic showing a representative system that includes
a logic device in which various aspects of the present invention may be
embodied. As will be understood by practitioners in the art from the
teachings provided herein, aspects of the invention are optionally
implemented in hardware and/or software. In some embodiments, different
aspects of the invention are implemented in either client-side logic or
server-side logic. As will be understood in the art, the invention or
components thereof may be embodied in a media program component (e.g., a
fixed media component) containing logic instructions and/or data that,
when loaded into an appropriately configured computing device, cause that
device to perform as desired. As will also be understood in the art, a
fixed media containing logic instructions may be delivered to a viewer on
a fixed media for physically loading into a viewer's computer or a fixed
media containing logic instructions may reside on a remote server that a
viewer accesses through a communication medium in order to download a
program component.

[0113] More specifically, FIG. 4 schematically illustrates computer 1000
to which mass spectrometer 1002 (e.g., an ESI-TOF mass spectrometer,
etc.), fluid transfer component 1004 (e.g., an automated mass
spectrometer sample injection needle or the like), and database 1008 are
operably connected. Optionally, one or more of these components are
operably connected to computer 1000 via a server (not shown in FIG. 4).
During operation, fluid transfer component 1004 typically transfers
reaction mixtures or components thereof (e.g., aliquots comprising
amplicons) from multi-well container 1006 to mass spectrometer 1002. Mass
spectrometer 1002 then detects molecular masses of the amplicons.
Computer 1000 then typically receives this molecular mass data,
calculates base compositions from this data, and compares it with entries
in database 1008 to identify species or strains of HPV in a given sample.
It will be apparent to one of skill in the art that one or more
components of the system schematically depicted in FIG. 4 are optionally
fabricated integral with one another (e.g., in the same housing).

[0114] While the present invention has been described with specificity in
accordance with certain of its embodiments, the following examples serve
only to illustrate the invention and are not intended to limit the same.
In order that the invention disclosed herein may be more efficiently
understood, examples are provided below. It should be understood that
these examples are for illustrative purposes only and are not to be
construed as limiting the invention in any manner.

Example 1

High-Throughput ESI-Mass Spectrometry Assay for the Identification of HPV

[0115] This example describes a HPV pathogen identification assay which
employs mass spectrometry determined base compositions for PCR amplicons
derived from HPV. The T5000 Biosensor System is a mass spectrometry based
universal biosensor that uses mass measurements to derived base
compositions of PCR amplicons to identify bioagents including, for
example, bacteria, fungi, viruses and protozoa (S. A. Hofstadler et. al.
Int. J. Mass Spectrom. (2005) 242:23-41, herein incorporated by
reference). For this HPV assay primers from Tables 1 and 2 may be
employed to generate PCR amplicons. The base composition of the PCR
amplicons can be determined and compared to a database of known HPV base
compositions to determine the identity of a HPV in a sample. Tables 1 and
2 shows exemplary primers pairs for detecting alphapapillomavirus,
betapapillomavirus, gammapapillomavirus, Mupapillomavirus, and
Nupapillomavirus. In Tables 1A and 2A, "I" represents inosine and
Tp=5-propynyluracil (also known as propynylated thymine).

[0116] It is noted that the primer pairs in Tables 1 and 2 could be
combined into a single panel for detection one or more HPV (e.g.,
multiple types of HPV). The primers and primer pairs of Tables 1 and 2
could be used, for example, to detect human and animal infections. These
primers and primer pairs may also be grouped (e.g., in panels or kits)
for multiplex detection of other bioagents such as flavivirus,
alphavirus, adenovirus, and other bioagents. In particular embodiments,
the primers are used in assays for testing product safety.

[0117] Because the molecular masses of the four natural nucleobases fall
within a narrow molecular mass range (A=313.058, G=329.052, C=289.046,
T=304.046, values in Daltons--See, Table 3), a source of ambiguity in
assignment of base composition may occur as follows: two nucleic acid
strands having different base composition may have a difference of about
1 Da when the base composition difference between the two strands is GA
(-15.994) combined with CT (+15.000). For example, one 99-mer nucleic
acid strand having a base composition of A27G30C21T21
has a theoretical molecular mass of 30779.058 while another 99-mer
nucleic acid strand having a base composition of
A26G31C22T20 has a theoretical molecular mass of
30780.052 is a molecular mass difference of only 0.994 Da. A 1 Da
difference in molecular mass may be within the experimental error of a
molecular mass measurement and thus, the relatively narrow molecular mass
range of the four natural nucleobases imposes an uncertainty factor in
this type of situation. One method for removing this theoretical 1 Da
uncertainty factor uses amplification of a nucleic acid with one
mass-tagged nucleobase and three natural nucleobases.

[0118] Addition of significant mass to one of the 4 nucleobases (dNTPs) in
an amplification reaction, or in the primers themselves, will result in a
significant difference in mass of the resulting amplicon (greater than 1
Da) arising from ambiguities such as the GA combined with CT event (Table
3). Thus, the same GA (-15.994) event combined with 5-Iodo-CT (-110.900)
event would result in a molecular mass difference of 126.894 Da. The
molecular mass of the base composition
A27G305-Iodo-C21T21 (33422.958) compared with
A26G315-Iodo-C22T20, (33549.852) provides a
theoretical molecular mass difference is +126.894. The experimental error
of a molecular mass measurement is not significant with regard to this
molecular mass difference. Furthermore, the only base composition
consistent with a measured molecular mass of the 99-mer nucleic acid is
A27G305-Iodo-C21T21. In contrast, the analogous
amplification without the mass tag has 18 possible base compositions.

[0119] Mass spectra of bioagent-identifying amplicons may be analyzed
using a maximum-likelihood processor, as is widely used in radar signal
processing. This processor first makes maximum likelihood estimates of
the input to the mass spectrometer for each primer by running matched
filters for each base composition aggregate on the input data. This
includes the response to a calibrant for each primer.

[0120] The algorithm emphasizes performance predictions culminating in
probability-of-detection versus probability-of-false-detection plots for
conditions involving complex backgrounds of naturally occurring organisms
and environmental contaminants. Matched filters consist of a priori
expectations of signal values given the set of primers used for each of
the bioagents. A genomic sequence database is used to define the mass
base count matched filters. The database contains the sequences of known
bioagents (e.g., types of HPV) and includes threat organisms as well as
benign background organisms. The latter is used to estimate and subtract
the spectral signature produced by the background organisms. A maximum
likelihood detection of known background organisms is implemented using
matched filters and a running-sum estimate of the noise covariance.
Background signal strengths are estimated and used along with the matched
filters to form signatures which are then subtracted. The maximum
likelihood process is applied to this "cleaned up" data in a similar
manner employing matched filters for the organisms and a running-sum
estimate of the noise-covariance for the cleaned up data.

[0121] The amplitudes of all base compositions of bioagent-identifying
amplicons for each primer are calibrated and a final maximum likelihood
amplitude estimate per organism is made based upon the multiple single
primer estimates. Models of system noise are factored into this two-stage
maximum likelihood calculation. The processor reports the number of
molecules of each base composition contained in the spectra. The quantity
of amplicon corresponding to the appropriate primer set is reported as
well as the quantities of primers remaining upon completion of the
amplification reaction.

[0122] Base count blurring may be carried out as follows. Electronic PCR
can be conducted on nucleotide sequences of the desired bioagents to
obtain the different expected base counts that could be obtained for each
primer pair. See for example, Schuler, Genome Res. 7:541-50, 1997; or the
e-PCR program available from National Center for Biotechnology
Information (NCBI, NIH, Bethesda, Md.). In one embodiment one or more
spreadsheets from a workbook comprising a plurality of spreadsheets may
be used (e.g., Microsoft Excel). First, in this example, there is a
worksheet with a name similar to the workbook name; this worksheet
contains the raw electronic PCR data. Second, there is a worksheet named
"filtered bioagents base count" that contains bioagent name and base
count; there is a separate record for each strain after removing
sequences that are not identified with a genus and species and removing
all sequences for bioagents with less than 10 strains. Third, there is a
worksheet, "Sheet1" that contains the frequency of substitutions,
insertions, or deletions for this primer pair. This data is generated by
first creating a pivot table from the data in the "filtered bioagents
base count" worksheet and then executing an Excel VBA macro. The macro
creates a table of differences in base counts for bioagents of the same
species, but different strains.

[0123] Application of an exemplary script, involves the user defining a
threshold that specifies the fraction of the strains that are represented
by the reference set of base counts for each bioagent. The reference set
of base counts for each bioagent may contain as many different base
counts as are needed to meet or exceed the threshold. The set of
reference base counts is defined by selecting the most abundant strain's
base type composition and adding it to the reference set, and then the
next most abundant strain's base type composition is added until the
threshold is met or exceeded.

[0124] For each base count not included in the reference base count set
for the bioagent of interest, the script then proceeds to determine the
manner in which the current base count differs from each of the base
counts in the reference set. This difference may be represented as a
combination of substitutions, Si=Xi, and insertions, Ii=Yi, or deletions,
Di=Zi. If there is more than one reference base count, then the reported
difference is chosen using rules that aim to minimize the number of
changes and, in instances with the same number of changes, minimize the
number of insertions or deletions. Therefore, the primary rule is to
identify the difference with the minimum sum (Xi+Yi) or (Xi+Zi), e.g.,
one insertion rather than two substitutions. If there are two or more
differences with the minimum sum, then the one that will be reported is
the one that contains the most substitutions.

[0125] Differences between a base count and a reference composition are
categorized as one, two, or more substitutions, one, two, or more
insertions, one, two, or more deletions, and combinations of
substitutions and insertions or deletions. The different classes of
nucleobase changes and their probabilities of occurrence have been
delineated in U.S. Patent Application Publication No. 2004209260 (U.S.
application Ser. No. 10/418,514) which is incorporated herein by
reference in entirety.

Example 3

Validation of Primer Pairs

[0126] The primer pairs were tested against a panel of papillomaviruses
obtained from ATCC. The following viruses were obtained as full-length
plasmid clones: ATCC 45150D (HPV-6b); ATCC 45151D (HPV-11); ATCC 45152D
(HPV-18); and ATCC 45113D (HPV-16). The broad primer pair number 2534
amplified all four viruses tested at two different dilutions of the
plasmids. A series of primer modifications, including, for example,
inosine substitutions to overcome potential sequence mismatches were
introduced into the forward and reverse primer pairs. Most of the
modified primers tested showed improved performance across the test
isolates. In addition to the primers broadly targeting the major species,
a series of primers targeting papillomavirus groups, A7, A9 and A10 that
account for over 30 different papillomaviruses were also tested. Table 2
provides the primer pairs used for papillomavirus identification and
indicates isolates tested, target virus groups and major species covered.

[0127] For additional testing and validation, two different HeLa cell
lines infected with HPV-18 were obtained from ATCC(CCL-2 and CCL-2.2).
These were tested at limiting dilutions using a subset of the primers
tested above. Results are shown below. The primer pairs used for this
test included the major human PaV primer pair 2685, the Group A7 targeted
primers 2544 and 2545 and the Group A10 primer 2546.

[0128] In addition to testing the performance of the primers on the cell
lines, plasmid DNA containing HPV-6b was spiked into the CCL-2 cell line
to determine the dynamic range of detection of the two viruses, cell line
derived HPV-18 and the plasmid-derived HPV-6b, simultaneously, In all the
tests done, the broad primers as well as the Group A7 primers showed
detection of HPV-18 in both cell lines at input levels between 1-10 cells
per well. At an estimated copy number of approximately 20 HPV-18 genomes
per cell, this corresponds to detection sensitivities between 20-200
genomes from cell lines containing papillomavirus sequences. In
experiments done with a co-spike of HPV-6b plasmid into these cell lines,
the detection ranges were comparable. HPV-6b was spiked in at two
different, fixed concentrations of 200 copies and 2000 copies per well
and amplified with the broad primer pair number 2534. Simultaneous
detection of HPV-6b and HPV-18 was observed when the plasmid DNA was
spiked in at 2000 copies into a range of CCL-2 cell concentration from
1000 to 0 per well. HPV-18 was detected in all wells with the exception
of the lowest input level (10 cells/well), in the presence of 2000 copies
of HPV-6b. HPV-6b (2000 copies) was detected in the presence of HeLa cell
loads up to 600 cells/well, with an effective HPV-18 concentration of
approximately 12000 genomes/well. In another experiment, a plasmid spike
of approximately 200 copies per well was used. In this case, HPV-18 was
detected at all test concentrations, including the lowest cell
concentration of 10 cells per well. The dynamic range for detection of
the two viruses simultaneously is between 5-10 fold at the lower and
higher ends, giving an overall dynamic range of approximately 25 fold for
the detection of competing templates in the presence of each other. These
experiments indicate that two or more viruses can be simultaneously
detected using the same assay.

Example 4

Testing of Primer Pairs Against Strains of Human Papillomaviruses

[0129] A series of human papillomavirus samples were tested using the
panel of primer pairs listed in Table 1 (primer pair numbers 2534, 2537,
2545, 2546, 2540, 2544, 2547 and 2684. The results are shown in Tables 5A
and 5B and include experimentally determined base compositions. Strains
of human papillomavirus identified are shown in the "Results" column. In
most cases, the experimentally-determined base compositions matched the
base compositions of strains of human papillomaviruses stored in a base
composition database.

[0130] Various modifications of the invention, in addition to those
described herein, will be apparent to those skilled in the art from the
foregoing description. Such modifications are also intended to fall
within the scope of the appended claims. Each reference (including, but
not limited to, journal articles, U.S. and non-U.S. patents, patent
application publications, international patent application publications,
gene bank accession numbers, interne web sites, and the like) cited in
the present application is incorporated herein by reference in its
entirety.